Methodological advances enabled by the construction of a synthetic yeast genome.

The international Synthetic Yeast Project (Sc2.0) aims to construct the first synthetic designer eukaryote genome. Over the past few years, the Sc2.

Emergence of fractal geometries in the evolution of a metabolic enzyme.

Fractals are patterns that are self-similar across multiple length-scales. Macroscopic fractals are common in nature; however, so far, molecular assembly into fractals is restricted to synthetic systems. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpiński triangles.

Getting to the point: unipolar growth of Hyphomicrobiales.

The governing principles and suites of genes for lateral elongation or incorporation of new cell wall material along the length of a rod-shaped cell are well described. In contrast, relatively little is known about unipolar elongation or incorporation of peptidoglycan at one end of the rod. Recent work in three related model systems of unipolar growth (Agrobacterium tumefaciens, Brucella abortus, and Sinorhizobium meliloti) has clearly established that unipolar growth in the Hyphomicrobiales order relies on a set of genes distinct from the canonical elongasome.

Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase.

The conversion of CO by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, methods following the microscopic details of CO conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO, water, bicarbonate, and other reactants with β-carbonic anhydrase from (CA) and crotonyl-CoA carboxylase/reductase from (Ccr), two of the fastest CO-converting enzymes in nature.

Ferredoxin reduction by hydrogen with iron functions as an evolutionary precursor of flavin-based electron bifurcation.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO and were chemolithoautotrophs that obtained their energy and electrons from H. The acetyl-CoA pathway of CO fixation is central to that view because of its antiquity: Among known CO fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H.

Polyketide Trimming Shapes Dihydroxynaphthalene-Melanin and Anthraquinone Pigments.

Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii are produced by a modular type II polyketide synthase system. A key enzyme involved in AQ biosynthesis is PlAntI, which catalyzes the hydrolysis of the bicyclic-intermediate-loaded acyl carrier protein, polyketide trimming, and assembly of the aromatic AQ scaffold.

Enhanced assembly of bacteriophage T7 produced in cell-free reactions under simulated microgravity.

On-demand biomanufacturing has the potential to improve healthcare and self-sufficiency during space missions. Cell-free transcription and translation reactions combined with DNA blueprints can produce promising therapeutics like bacteriophages and virus-like particles. However, how space conditions affect the synthesis and self-assembly of such complex multi-protein structures is unknown.

In Silico Design, In Vitro Construction, and In Vivo Application of Synthetic Small Regulatory RNAs in Bacteria.

Small regulatory RNAs (sRNAs) are short non-coding RNAs in bacteria capable of post-transcriptional regulation. sRNAs have recently gained attention as tools in basic and applied sciences, for example, to fine-tune genetic circuits or biotechnological processes. Even though sRNAs often have a rather simple and modular structure, the design of functional synthetic sRNAs is not necessarily trivial.

A MoClo-Compatible Toolbox of ECF Sigma Factor-Based Regulatory Switches for Proteobacterial Chassis.

The construction of complex synthetic gene circuits with predetermined and reliable output depends on orthogonal regulatory parts that do not inadvertently interfere with the host machinery or with other circuit components. Previously, extracytoplasmic function sigma factors (ECFs), a diverse group of alternative sigma factors with distinct promoter specificities, were shown to have great potential as context-independent regulators, but so far, they have only been used in a few model species. Here, we show that the alphaproteobacterium , which has been proposed as a plant-associated bacterial chassis for synthetic biology, has a similar phylogenetic ECF acceptance range as the gammaproteobacterium .

Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in .

Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated.

Structure of the GDP-bound state of the SRP GTPase FlhF.

The GTPase FlhF, a signal recognition particle (SRP)-type enzyme, is pivotal for spatial-numerical control and bacterial flagella assembly across diverse species, including pathogens. This study presents the X-ray structure of FlhF in its GDP-bound state at a resolution of 2.28 Å.

Enlightening epigenetics: optochemical tools illuminate the path.

Optochemical tools have become potent instruments for understanding biological processes at the molecular level, and the past decade has witnessed their use in epigenetics and epitranscriptomics (also known as RNA epigenetics) for deciphering gene expression regulation. By using photoresponsive molecules such as photoswitches and photocages, researchers can achieve precise control over when and where specific events occur. Therefore, these are invaluable for studying both histone and nucleotide modifications and exploring disease-related mechanisms.

A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides.

Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level.

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, abbreviated as SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield.

A dynamic bactofilin cytoskeleton cooperates with an M23 endopeptidase to control bacterial morphogenesis.

Bactofilins have emerged as a widespread family of cytoskeletal proteins with important roles in bacterial morphogenesis, but their precise mode of action is still incompletely understood. In this study, we identify the bactofilin cytoskeleton as a key regulator of cell growth in the stalked budding alphaproteobacterium . We show that, in this species, bactofilin polymers localize dynamically to the stalk base and the bud neck, with their absence leading to unconstrained growth of the stalk and bud compartments, indicating a central role in the spatial regulation of cell wall biosynthesis.

The dual GGDEF/EAL domain enzyme PA0285 is a Pseudomonas species housekeeping phosphodiesterase regulating early attachment and biofilm architecture.

Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclases (DGCs) activity favor biofilm formation, while low levels were maintained by phosphodiesterases (PDE) encourage planktonic lifestyle.

The enigmatic epitranscriptome of bacteriophages: putative RNA modifications in viral infections.

RNA modifications play essential roles in modulating RNA function, stability, and fate across all kingdoms of life. The entirety of the RNA modifications within a cell is defined as the epitranscriptome. While eukaryotic RNA modifications are intensively studied, understanding bacterial RNA modifications remains limited, and knowledge about bacteriophage RNA modifications is almost nonexistent.

An Integrated Affinity Chromatography-Based Approach to Unravel the sRNA Interactome in Nitrogen-Fixing Rhizobia.

The activity mechanism and function of bacterial base-pairing small non-coding RNA regulators (sRNAs) are largely shaped by their main interacting cellular partners, i.e., proteins and mRNAs.

The virulence regulator VirB from Shigella flexneri uses a CTP-dependent switch mechanism to activate gene expression.

The transcriptional antisilencer VirB acts as a master regulator of virulence gene expression in the human pathogen Shigella flexneri. It binds DNA sequences (virS) upstream of VirB-dependent promoters and counteracts their silencing by the nucleoid-organizing protein H-NS. However, its precise mode of action remains unclear.

Manipulation of GameXPeptide synthetase gene expression by a promoter exchange alters the virulence of an entomopathogenic bacterium, , by modulating insect immune responses.

An entomopathogenic bacterium, subsp. , is mutualistic to its host nematode, . The infective juvenile nematodes enter target insects through natural openings and release the symbiotic bacteria into the insect hemocoel.

Creating new-to-nature carbon fixation: A guide.

Synthetic biology aims at designing new biological functions from first principles. These new designs allow to expand the natural solution space and overcome the limitations of naturally evolved systems. One example is synthetic CO-fixation pathways that promise to provide more efficient ways for the capture and conversion of CO than natural pathways, such as the Calvin Benson Bassham (CBB) cycle of photosynthesis.